Chromatographic apparatus

ABSTRACT

Chromatographic apparatus for developing circular chromatograms on the sample spotted stationary layer of a TLC plate including: a base providing a pattern of solvent wells corresponding to sample positions facing the TLC plate, a wick holding plate between the base and the TLC plate, a sealing layer between the base and the wick holding plate with an aperture registering with each solvent well, wicks extending through the wick holding plate each with one end dipping in a well and the other end contacting a sample on the adsorbent surface, a spacing and sealing layer between the wick holder and the TLC plate with an aperture whose periphery surrounds each operative wick to provide a development chamber bounded by portions of each plate and such periphery, and means for clamping the assembly together to seal the chambers and assure contact of the wicks with the samples so that they will transport solvents from the wells to the samples.

' United States Patent [191 Kremer Dec. 23, 1975 [75] Inventor:

[ CHROMATOGRAPHIC APPARATUS Richard D. Kremer, Keene, NH.

[73] Assignee: Schleicher & Schuell, Incorporated,

Keene, NH.

[22] Filed: Mar. 27, 1975 A [21] Appl. No.: 562,490

52 US. Cl 210/198 c [51] Int. Cl. B01D 15/08 [58] Field of Search 210/31 C, 198 C 56] p References Cited UNITED STATES PATENTS 2,986,280 5/1961 Magnuson et al 210/198 C 3,738,493 6/1973 Cummins et al. 210/198 C 3,757,952

9/1973 Baitsholts et al 210/198 C Primary Examiner-John Adee [57] ABSTRACT Chromatographic apparatus for developing circular chromatograms on the sample spotted stationary layer of a TLC plate including: a base providing a pattern of solvent wells corresponding to sample positions facing the TLC plate, a wick holding plate between the base and the TLC plate, a sealing layer between the base and the wick holding plate with an aperture registering with each solvent well, wicks extending through the wick holding plate each with one end dipping in a well and the other end contacting a sample on the adsorbent surface, a spacing and sealing layer between the wick holder and the TLC plate with an aperture whose periphery surrounds each operative wick to provide a development chamber bounded by portions of each plate and such periphery, and means for clamping the assembly together to seal the chambers and assure contact of the wicks with the samples so that they will transport solvents from the wells to the samples.

6 Claims, 5 Drawing Figures U... atent Dec.23, 1975 Sheet20f3 3,928,203

US. Patent Dec. 23, 1975 Sheet 3 of3 3,928,203

FIG 5 FIG 4 appropriate phases to be used, many chromatographers I employ hand or commercially coated microscope sl|des, containing various stationary phases, comprising various solid adsorbents'or liquid partitioning or reverse phase partitioning agentsJEach ofthese slides in turn is placed in a small individual chamber containing a particular mobile phase, usually chosen from one of several suggested eluotropic series (series of solvents listed in the order of their increasing elutive power). An example of such a series is:

. Benzene I g Methylene dichloride l Chloroform I 9. Diethyl ether,

l0. .Ethyl acetate 11. Pyridine l2. Acetone l3. n-Propanol l. Petroleum ether 2. Cyclohexane 3. Carbon tetrachloride 4. Trichloroethylene 5. Toluene l4. Ethanol l5. Methanol .16. Water Based on the results of development in several such chambers, appropriate mobile phases (solvents) are selected for further work.

Circular chromatography is a particularly useful mobile phase selection tool because of its superior resolving power within a small development distance and time-span.

Stahl has shown (Chemiken-Ztg. 82, 323 (1958), Parfumerie U. Kosm. 39, 564 (1958)) that the microcircular technique is excellently suited for quick determination of the requisite solvent. On pages 73 and 74 of his text Thin. Layer Chromatography, 2d Ed, published by Springer-Verlag New York lnc., 1969,

Stahl describes three devices for carrying out this tech-* nique: the standing :pipette and two simple circular chambers. l

However, as the system employing'a standing pipette is open, it has the disadvantage'tha-t the solvent rapidly evaporates, and the chromatograms produced are therefore quite small, 1 to 2 cm'in'diameter. Another The two circular chamber designs solve the problem of evaporation by, utilizinga closed chamber system. But, as with the microscope slide technique mentioned above, only one solvent system can be evaluated by each chamber. This necessitates the use of a separate chamber for each solvent to be evaluated, and requires considerable time forpreparation and clean up of such multiple apparatus. The developed chromatographs are all on separate plates and must be assembled and arrangedv systematically for purposes of comparison and evaluation. In addition, these chambers utilize a chromatographic plate which has been perforated from front to back to create a 2 mm diameter channel to accommodate a wick and/or allow solvent'flow to the stationary layer on the plate.

SHORT STATEMENT OF 'THE lNVENTlON An object of the present invention is to'provide a chromatographic apparatus for the development of circular chromatograms that is simple in construction, compact, easy to use, and permits rapid evaluation of up to at least sixteen mobile phases (solvents) or samples, utilizing standard 20 X 20 cm chromatographic plates (TLC plates). a

It is also an object to provide such a system that is economical in its use of solvent.

The apparatus of this invention does not require any subdivision of the stationary layer (i.e., adsorbent surface), as by scoring or other adsorbent removal, to create isolated channels or zones, as is normally done when different mobile phases are run simultaneously on a single plate.

In addition, the system of the invention may be quickly and easily set up for use, and, in particular, eliminates the time required to thread flexiblewicking through wicking support means, which is a laborious and time-consuming operation. Y

The system of the invention also provides for simple selection of the number and size of developed chromatograms to be obtained.

With the foregoing in mind, the invention is featured by the provision of chromatographic apparatus for developing chromatograms on the stationary layer of a TLC plate spotted at predetermined samplepositions with one or more samples, the apparatus including a base providing solvent wells in a predetermined pattern, each open at the upper surface of the base so as to face the stationary layer of the TLC plate when ;the latter is positioned in the apparatus. Selection means are provided adapted tob'e held between thebase and the TLC plate to'select solvent wells corresponding in number and position to the positions of the samples. The selection meanscomprises a wick holding plate arranged to'be .held between the base and the TLC plate, having holes corresponding both in number and position to the aforesaid sample positions and corresponding wellsithe' plate being adapted to hold wicks in said holes,'each wick having a solvent take-up portion for dipping in a well and a sample contacting portion for contacting a sample;and a spacing and sealing layer between the wick holding plate and the TLC'plate, such layer-including a spacingaperture having a periphery surrounding each sample contacting portion and spaced therefrom to define a development chamber bounded by portions of the last named plates and the said periphery. Finally, pressure means are arranged to urge theplates together to seal eaeh development chamberahtl t6 maintain the TLC plate in developing contact with each wick sample contacting portion, whereby each wick will transport solvent from its solvent well through its development chamber to a sample for development thereof.

A further feature is the provision of alternative selection means which can be substituted in the assembly for the selection means just described and comprising a second wick holding plate and a second spacing and sealing layer with a different number and arrangement of matching holes and apertures to correspond with samples spotted at other positions on the stationary layer of the TLC plate and their corresponding wells.

The base preferably is provided with three upstanding wall portions to receive and hold the plates and associated spacing and sealing felts in proper position and the pressure means preferably comprises upstanding studs mounted on the base and associated pressure bars and threaded knobs coacting with the studs to clamp the assembly together by means of the pressure bars.

A desirable further feature is the provision of a spacing and solvent isolating felt between the base and the wick holding plate provided with suitable apertures corresponding with the solvent wells in use so as to reduce evaporation and prevent spread of solvent vapor from one well area to another.

Further objects, features and advantages will become apparent from the following description of a preferred embodiment taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS In the drawings, illustrating a preferred embodiment of the invention,

FIG. 1 is a longitudinal cross-sectional view of the assembled apparatus preparatory to clamping the parts together;

FIG. 2 is a cross-sectional view taken on line 22 of FIG. 1;

FIG. 3 is an exploded view in perspective showing the several components preparatory to assembly;

FIG. 4 is an exploded view of certain alternative components which may be used in the assembly to produce larger sized chromatograms; and

FIG. 5 is an elevation on an enlarged scale of a solvent transfer wick usable in the novel apparatus.

DESCRIPTION OF PREFERRED EMBODIMENT Referring to the drawings, the components of a preferred embodiment of the chromatographic development apparatus of the invention are shown in the exploded views of FIGS. 3 and 4. The system comprises a base plate (FIG. 3) providing three sets of solvent wells, all open at the upper surface of the base plate. There are 16 solvent wells 12, each one-halfinch (12.7 mm) in diameter, four solvent wells 14, each threequarters (19.04 mm) of an inch in diameter, and one central solvent well 16 that is 1 inch (25,38 mm) in diameter.

Base 10 further provides walls 50, 52 and 54 on three edges of the base, extending above the upper surface. The fourth edge 56 does not provide a wall but is open to permit other elements of the system to be slid into position and retained within walls 50, 52 and,54. Exterior to each of the opposed walls 50 and 54 and attached to the base, by projections are two upstanding threaded studs 34 external to the base plate.

The chromatographic development system further comprises "a base plate sealer felt 18 that covers the upper surface of base plate 10 and provides apertures 19 registering with wells 12, 14 and I6. Felt I8 acts as a solvent isolating layer which reduces evaporation, and prevents mixing of the vapors from the solvents contained in the various wells.

Not all the solvent wells in base 10 are employed at the same time. The system of the invention makes possible the development of one chromatogram having a diameter of up to mm, or in the alternative, from one to four chromatograms having diameters of up to 87 mm or, as a third alternative, from I to 16 chromatograms having diameters of up to 40 mm. The selection of one of these alternatives is made possible by the use of selection means, comprising one of the alternative wick holding plates combined with one of the alternative spacer felts, to be described.

As seen in FIG. 3, one form of wick holding plate 22 provides 16 wick holes 23 corresponding with the 16 half-inch (12.7 mm) diameter solvent wells 12 in base plate 10. 16 wicks 24 are inserted in wick holding plate 22 through holes 23. In FIG. 3, three of the wicks are not shown, in order to show the wick holes. In addition, plate 22 provides three registration holes 37, placed asymmetrically with respect to the edges of the plate, whose function is explained below.

A wick (FIG. 5) suitable for use in apparatus of the invention is molded of a porous solvent resistant wicking material, such as polyethylene or polypropylene. Other solvent resistant plastics, fibrous materials and ceramics could also be employed, but polyethylene and polypropylene are particularly suitable because they are commercially available, inexpensive, and provide firm body construction. The particular wick employed in the specific embodiment herein described is manu factured by Porex Materials Corp. for use in pens.

Each wick 24, seen in detail in' FIG. 5, has an elongated cylindrical body portion 25 about 1/16 inch (1.85 mm) thick, including a solvent takeup base portion 27. The wick further has a rounded head 29 about 1.50 mm long which acts as a sample contact portion and which includes a retaining edge 33 of larger diameter than body portion 25. The wick is about inch (18.0 mm) long overall.

A first alternative spacer and sealer felt 26 provides sixteen circular holes 36 in diameter 40 mm. The felt thickness is approximately equal to the length of wick head 29, i.e., of the order of 2.60 mm. When the spacer felt is placed on wick holding plate 22, the periphery of each spacer felt hole 36 surrounds one of the sixteen wick heads 29 protruding above plate 22. A thin-layer chromatography (TLC) plate 28 provides a stationary layer 30 which has been sample spotted at locations indicated by reference numeral 35.

The removable pressure bars 32 are provided with openings 53 spaced to receive the four upstanding studs 34 fixed to base 10. Four threaded fastening knobs 55 are provided to engage the protruding ends of posts 34, in orderto press TLC plate 28 downwardly toward base 10, to insure uniform good contact of wick points 31 against sample spots 35 and to insure sealing contact of the plates with the felts.

An alternative wick holding plate 38 (FIG. 4) provides four wick holes 40 and one central wick hole 42. The four wick holes 40 correspond with the 4% inch (19.04 mm) diameter wells 14 in base plate 10, while the central wick hole 4.2 corresponds with the single 1-inch (25.38 mm) diameter well 16 in'base plate 10. In addition, plate 38 provides three registration holes 39, asymmetrically placed with respect to the edges of the plate. Two further spacer felts 44 and 46 are pro- I vided for use alternatively with plate 38; felt 44 provides four holes 45 each of diameter of 87 mm, and felt 46 provides a single hole 47 having a diameter of 170 When wick holding plate 22, providing ,up to' 16 wicks, is employed together with spacer felt 26, any or all of the sixteen /2 (12.7 mm) solvent wells 12 in base plate may be selected for use in developing as many as sixteen 40 mm diameter chromatograms on TLC plate 28.

When wick holding plate 38 is employed with spacer felt 44, with up to four wicks disposed in the fourholes 40, any or all of the four inch (19.04 mm) holes 14 in base plate 10 are selected, permitting development of as many as four chromatograms up to 87 mm in diameter. When wick holding plate 38 is employed with spacer felt 46, with a single wick provided in central hole 42, the central l-inch (25.38 mm) well 16 in base plate 10 is selected permitting development of a single chromatogram up to 170 mm in diameter.

In use, the base plate sealer felt 18 is laid inside the side walls of the base plate 10, so that the apertures 19 in the sealer felt and the solvent wells 12, 14 and 16 in the base plate are aligned with one another. A standard 20 X 20 cm TLC plate, such as plate 28, is then laid on top of the sealer felt, stationary layer side up. On top of this is placed a selected one of the two wick holding plates 22 and 38, the choice being determined by the number and size of the developed chromatograms desired by the analyst. With a soft lead pencil, using the wick holes (23, 40, or 42) and registration holes (37 or 39) as guides, the TLC plate is marked through the wick holes for sample spotting, and registration marks are made through the registration holes. The marked TLC plate and the wicking plate are then removed from the base plate and the desired samples are then spotted on the TLC plate at the marked locations.

Wicks 24 are then inserted into the appropriate holes in the selected wicking plate. The appropriate spacer felt (26, 44 or 46) is laid on top of the wicking plate and aligned therewith. Next, the samplespotted TLC plate 28 is laid on top of the spacer felt, this time with the stationary layer side 30 down, and aligned with the wick holding plate by means of the registration marks and the corresponding registration holes in the wick holding plate. The spotted samples are thereby positioned over the wicks. Once positioned, the wick holding plate, spacer felt and TLC plate are conveniently taped together as by tape 58 (FIGS. 1 and 2) to form a layered sandwich (as indicated at 20, in FIG. 3), which may be easily handled without disturbing the alignment of the wicks and spotted samples. The wick holding plate and spacer felt serve as selection means to select the appropriate set of solvent wells.

A solvent application plate (not shown) having small apertures centered over the base plate wells, may be used during filling of the solvent wells, to reduce losses of volatile solvents by evaporation. The appropriate solvent wells in base plate 10 are filled by means of a dropper, micropipette, syringe, or other suitable means. Each /z-inch (12.7 mm) well 12 requires about 0.25 ml of solvent for development of a mm diameter circle. Each inch (19.04 mm) well 14 requires about 1.25 ml of solvent for development of a 87 mm diameter circle. Combinations of solvent may be conveniently and easily made, using a dropper or pipette.

After the solvent wells have been filled, layered sandwich 20, previously prepared as described, is aligned with and laid on top of the base plate sealer felt 18, so that the 'takeup ends 27 of the wicks 24, protruding below the wick holding plate, dip into the filled solvent wells. The pressure bars 32 are placed in position by sliding them down over the studs 34 until they are in contact with the glass (uncoated) upper surface of the TLC plate, which forms the top side of the layered sandwich. The pressure bars 34 are secured in place by means of threaded fastening knobs 55, which are screwed down on the tops of the studs. The pressure thus exerted on TLC plate 28 by the bars assists in bringing all of the wick heads 29 into firm contact with the stationary layer 30 of the TLC plate. It also presses the selected spacer layer (26, 44 or 46) into firm contact with both the wicking plate and the TLC plate. Thus, a tightly sealed cyclindrical development chamber is formed by the wicking plate, the periphery of the spacer felt aperture, and the TLC plate around each sample spot with the wick head in contact therewith. Each such chamber is positioned above its individual solvent feed system and is isolated from all other such solvent feed systems. The atmosphere in each of these chambers will rapidly become saturated with the vapor of the particular solvent with which the wick is in contact. Thus equilibrium between the liquid and gaseous phases is quickly established.

The solvent is drawn up the wick by capillary action and comes into contact with the spotted sample on the stationary layer of the TLC plate, and chromatographic development begins. Development may be limited by limiting the quantity of solvent with which the well is initially filled, or the analyst may remove the sandwich package from the base plate in order to stop development when the solvent front reaches the edge of the hole in the spacer felt. The TLC plate is then separated from the sandwich package and dried, and thereafter is visualized in the conventional manner.

Using the set of sixteen /z-inch (12.7 mm) wells 12 in combination with selection means comprising spacer felt 26 and wick holding plate 22, an entire eluotropic series can be run at one time. The series proposed by Stahl (Thin-Layer Chromatography, 2 Ed., Springer- Verlag New York Inc. 1969, p. 202) contains only 13 solvents; Truter (Thin Film Chromatography, lnterscience, 1963, p. 29) lists 14. Once the initial series has been run, a selected portion of a more extensive series can be used (Chromatography, 2 Ed., E. Heftmann, p. 59), or multiple systems can be tried, made up of those solvents which showed promise during the initial run. By using a single 20 X 20 cm plate in this manner, it is possible to eliminate the use of a larger number of individual tank type chambers, and to cut down on preparation time and cleanup time. Visualization of the chromatograms by ultraviolet light, reagent spraying, or the like is also facilitated because the chromatograms are all contained on one plate and can be visualized as a unit. Also, the finished chromatograms may be displayed compactly and in logical order, to facilitate comparison and evaluation of results.

When it is desired to develop larger size chromatograms on the same type of standard size TLC plate, alternative wick holder 38 may be employed and, with it, either of the two spacer felts 44 and 46. Where four wicks are used up to four chromatograms up to 87 mm in diameter may be developed; with a single central wick and felt 46, a single chromatogram up to 170 mm in diameter may be developed.

While the invention has particular utility as applied to the utilization of standard sized TLC plates of X 20 cm, it will be understood that the scope of the inven tion is not so limited and its advantages may be enjoyed using components of any suitable size.

What is claimed is:

l. Chromatographic apparatus for developing chromatograms on the stationary layer of a TLC plate spotted at predetermined sample positions with one or more samples, said apparatus comprising a base providing solvent wells in a predetermined pattern, each open at the upper surface of the base so to face the adsorbent surface of the TLC plate when the latter is positioned in the apparatus,

selection means adapted to be held between said base and said TLC plate to select solvent wells corresponding in number and position to the positions of said samples, said means comprising a wick holding plate arranged to be held between said base and said TLC plate and having holes corresponding both in number and position to the aforesaid sample positions and corresponding wells,

said plate being adapted to hold wicks in said holes, each wick having a solvent take-up portion for dipping in a well and a sample contacting portion for contacting a sample, and

a spacing and sealing layer between said wick holding plate and said TLC plate, said layer including a spacing aperture having a periphery surrounding each said sample contacting portion and spaced therefrom to define a development chamber bounded by portions of said last named plates and said periphery, and

pressure means arranged to urge said plates together to seal each said chamber and to maintain said TLC plate in developing contact with each said wick sample contacting portion,

whereby each wick will transport solvent from its solvent well through its development chamber to a sample for development thereof.

2. The combination of claim 1 wherein said base is provided with wall portions extending above said base upper surface, for receiving and retaining said selection means and said TLC plate.

3. The combination of claim 1, further including a solvent isolating layer between said base upper surface and said wick holding plate, said isolating layer including an aperture registering with each solvent well opening.

4. The combination of claim 1, wherein said pressure means comprises a pair of studs extending upwardly on opposite sides of said base,

a removable pressure bar having mounting holes to receive said studs. and

threaded knobs engaging said studs.

5. A chromatography system comprising a base including at least one solvent well open at the upper surface of said base a wick holding apertured plate above said base at least one wick extending from a solvent well through an aperture of said wick holding plate and extending thereabove a spacing and sealing layer above said wick holding plate and having an aperture having a periphery surrounding each wick a TLC plate above said spacing layer and having a stationary layer adjacent said spacing layer, said TLC plate being adapted to be spotted with a sample to register with each wick said wick holding plate, said spacing aperture periphery and said TLC plate together defining a generally closed development chamber I whereby a solvent contained in said base solvent well is transported by said wick through said develop ment chamber to said sample spotted TLC plate for and their corresponding solvent wells. 

1. CHROMATOGRAPHIC APPARATUS FOR DEVELOPING CHROMATOGRAMS ON THE STATIONARY LAYER OF A TLC PLATE SPOTTED AT PREDETERMINED SAMPLE POSITIONS WITH ONE OR MORE SAMPLES, SAID APPARATUS COMPRISING A BASE PROVIDING SOLVENT WELLS IN A PREDETERMINED PATTERN, EACH OPEN AT THE UPPER SURFACE OF THE BASE SO AS TO FACE THE ADSORBENT SURFACE OF THE TLC PLATE WHEN THE LATER IS POSITIONED IN THE APPARATUS, SELECTION MEANS ADAPTED TO THE HELD BETWEEN SAID BASE AND SAID TLC PLATE TO SELECT SOLVENT WELLS CORRESPONDING IN NUMBER AND POSITION TO THE POSITIONS OF SAID SAMPLES, SAID MEANS COMPRISING A WICK HOLDING PLATE ARRANGED TO BE HELD BETWEEN SAID BASE AND SAID TLC PLATE AND HAVING HOLES CORRESPONDING BOTH IN NUMBER POSITION TO THE AFORESAID SAMPLE POSITIONS AND CORRESPONDING WELLS, SAID PLATE BEING ADAPTED TO HOLD WICKS IN SAID HOLES, EACH WICK HAVING A SOLVENT TAKE-UP PORTION FOR DIPPING IN A WELL AND A SAMPLE CONTACTING PORTION FOR CONTACTING A SAMPLE, AND A SPACING AND SEALING LAYER BETWEEN SAID WICK HOLDING PLATE AND SAID TLC PLATE, SAID LAYER INCLUDING A SPACING APERTURE HAVING A PERIPHERY SURROUNDING EACH SAID SAMPLE CONTACTING PORTION AND SPACED THEREFROM TP DEFINE A DEVELOPMENT CHAMBER BOUNDED BY PORTIONS OF SAID LAST NAMED PLATES AND SAID PERIPHERY, AND PRESSURE MEANS ARRANGED TO URGE SAID PLATES TOGETHER TO SEAL EACH SAID CHAMBER AND TO MAINTAIN SAID TLC PLATE IN DEVELOPING CONTACT WITH EACH SAID WICK SAMPLE CONTACTING PORTION, WHEREBY EACH WICK WILL TRANSPORT SOLVENT FROM ITS SOLVENT WELL THROUGH ITS DEVELOPMENT CHAMBER TO A SAMPLE FOR DEVELOPMENT THEREOF.
 2. The combination of claim 1 wherein said base is provided with wall portions extending above said base upper surface, for receiving and retaining said selection means and said TLC plate.
 3. The combination of claim 1, further including a solvent isolating layer between said base upper surface and said wick holding plate, said isolating layer including an aperture registering with each solvent well opening.
 4. The combination of claim 1, wherein said pressure means comprises a pair of studs extending upwardly on opposite sides of said base, a removable pressure bar having mounting holes to receive said studs, and threaded knobs engaging said studs.
 5. A chromatography system comprising a base including at least one solvent well open at the upper surface of said base a wick holding apertured plate above said base at least one wick extending from a solvent well through an aperture of said wick holding plate and extending thereabove a spacing and sealing layer above said wick holding plate and having an aperture having a periphery surrounding each wick a TLC plate above said spacing layer and having a stationary layer adjacent said spacing layer, said TLC plate being adapted to be spotted with a sample to register with each wick said wick holding plate, said spacing aperture periphery and said TLC plate together defining a generally closed development chamber whereby a solvent contained in said base solvent well is transported by said wick through said development chamber to said sample spotted TLC plate for development of said sample.
 6. The combination of claim 1 wherein alternative selection means are provided to be substituted for the first named selection means, said alternative selection means comprising a second wick holding plate and a second spacing and sealing layer having matching holes and apertures arranged in an different pattern from those of the first named selection means so as to correspond with other sample positions on said stationary layer and their corresponding solvent wells. 